The mammary gland is a complex organ whose growth and development are controlled by the interaction of a wide variety of hormones and growth factors also involved in the etiology and progression of the cancerous state. Our emphasis has been on the interactions of prolactin (PRL), estrogen (E), and progesterone (P) during the peripubertal period and the lobulo-alveolar development of pregnancy as well as during tumorigenesis. We have shown that E and P are required to promote development of the primary/secondary ductal network in addition to other endocrine growth factor(s), and that P facilitates the formation of tertiary side-branches. The Hox-related homeobox containing gene, Msx2, is highly expressed during branching morphogenesis where our studies in vivo and in vitro showed that its expression is regulated by P in the presence of E. Concurrent with these morphological changes, progesterone receptor (PR) localizes at early branch points. During peripubertal morphogenesis PR distribution shifted from a homogeneous to a heterogeneous pattern. Concomitantly the PRL receptor (PRLR) undergoes a similar shift in pattern. We demonstrated differential transcription of the four PRLR isoforms by stromal as well as epithelial cells throughout development. The distribution of the PRLR in the epithelium, like that of the PR, progressed from a homogeneous to a heterogeneous pattern. Hence, while exogenous P or PRL alone was without effect on epithelial proliferation in ovariectomized mice, these hormones synergize to stimulate epithelial and stromal proliferation. The role of P, in part is to regulate the PRL receptor. We are studying changes in the vascular network that facilitates lactogenesis and tumorigenesis in the mammary gland. Our data support the conclusion that specific cell types within the mammary gland differentially transcribe VEGF and that it functions as an autocrine/paracrine endothelial growth factor under hormonal regulation. We have identified that PRL induces expression of VEGF in breast cells through an increase in the transcription factor Egr-1. Additional studies aim to understand the role of PRL in the etiology and progression of human breast cancer. Specifically, we are examining the role of PRLR isoforms and autocrine/paracrine PRL in tumorigenesis and carcinogenic susceptibility. Comparisons between cancerous and adjacent, noninvolved tissue from the same breast of 23 patients showed that, on average, both PRL and PRLR mRNA expression was significantly higher in the cancerous tissue compared to the noninvolved tissue. The various forms of the PRLR differ in their cytoplasmic domains due to alternate splicing. Specific antibodies to the various isoforms are under development. A third novel isoform (delta7/11) encodes a protein capable of being secreted as a soluble PRL-binding protein in cell culture. Analysis of tumorous tissue and normal tissue from the same patient failed to detect a difference in the expression of the soluble PRLR isoform and no protein was detected in serum. Functional analyses indicated that hPRLR-SF1b is a strong dominant negative to the differentiative function of the PRLR long form while hPRLR-SF1a is a weaker dominant negative. Differential abundance of SF1a, SF1b and delta7/11 expression was detected in normal breast, colon, placenta, kidney, liver, ovary and pancreas, and breast and colon tumors. Expression of SF1a, but not SF1b or LF was induced in prostate cancer cells in culture exposed to the mutant human PRL, S179DPRL. In the mouse, the short forms o the receptor act as dominant negatives of the differentiative function of PRL in part by inhibiting the synthesis of the LF. Taken together, these data indicate the presence of multiple isoforms of the hPRLR that may function to modulate the endocrine and autocrine effects of PRL in normal human tissue and cancer.